Technologies for Multiple Pathogen Detection

Transcription

1 Technologies for Multiple Pathogen Detection Patrick Tang, MD, PhD, FRCPC Medical Microbiologist, BCCDC Public Health Microbiology & Reference Laboratory Clinical Assistant Professor, UBC

2 Technologies for Multiple Pathogen Detection Molecular diagnostic technologies Challenges of multiplex pathogen detection Future of molecular microbiology

3 BCCDC Public Health Microbiology & Reference Laboratory

4 BCCDC Public Health Microbiology & Reference Laboratory

5 Limitations of Traditional Methods Speed Sensitivity Taxonomy-based Spectrum of targets Specialized training

6 Ideal Microbiology Test Rapid Sensitive and specific Syndrome-based multiplexing Shared technology platform Common laboratory training

7

8 Detection of Host Response Serology (Host Immune Response)

9 Detection of Pathogen Microscopy Culture Antigens Nucleic Acids

10

11 Nucleic Acid Detection Non-amplified Nucleic Acid Probe labeled DNA or RNA probe (enzyme, fluorescence, etc.) Signal Amplification increase concentration of labeled molecules attached to target Target Amplification enzyme-mediated synthesis of copies of the target nucleic acid Probe Amplification amplification products generated only from probes Metagenomics (sequencing)

12 Non-Amplified Nucleic Acid Probe Liquid-phase hybridization protection assay (Gen-Probe) Single-stranded DNA probe labeled with acridinium ester is added to sample If the probe binds to its complementary target sequence, the acridiniumester is protected from alkaline hydrolysis otherwise, acridinium ester will be hydrolyzed Acridiniumester emits light upon addition of peroxides DNA probe target sequence

13 Quantum Dots Semiconductor nanocrystals Quantum confinement of electrons Shape and size determine fluorescence spectrum 100 times brighter than regular fluorescent report dyes

14 GeneFluidics Electrochemical detection Capture probe is bound to sensor surface HRP-linked detection probe binds to second site on target DNA HRP reaction generates current proportional to target concentration HRP e-

15 Signal Amplification Branched DNA (Siemens) sandwich hybridization assay with multiple sets of probes bdnahas 15 identical branches, each can bind 3 labeled probes bdna enzyme-labeled probes target sequence target probes capture probes microwell with capture probes

16 Microarray-based platform Gold nanoparticle detection Signal amplification through deposition of elemental silver onto gold nanoparticles Measure light scatter from nanoparticles Nanosphere

17 Target Amplification Polymerase Chain Reaction (PCR) Transcription-mediated amplification (TMA) / Nucleic acid sequence-based amplification (NASBA) Strand displacement amplification (SDA) plus many other strategies

18 Multiplexing

19 Multiplex PCR Electrophoresis Fluorescent probes (FRET) Hybridization Microarrays Bead-based arrays Mass spectroscopy detection Sequencing Spatial multiplexing

20 Multiplex PCR Electrophoresis Fluorescent probes (FRET) Hybridization Microarrays Bead-based arrays Mass spectroscopy detection Sequencing R Spatial multiplexing Molecular Beacon TaqMan 5 Exonuclease Probe Q

21 Multiplex PCR Electrophoresis Fluorescent probes (FRET) Hybridization Microarrays Bead-based arrays Mass spectroscopy detection Sequencing Spatial multiplexing Line Probe Assays

22 Multiplex PCR Electrophoresis Fluorescent probes (FRET) Hybridization Microarrays Bead-based arrays Mass spectroscopy detection Sequencing Spatial multiplexing AutoGenomics INFINITI Akonni TruArray

23 Multiplex PCR Electrophoresis Fluorescent probes (FRET) Hybridization Microarrays Bead-based arrays Mass spectroscopy detection Sequencing Spatial multiplexing Luminex

24 Multiplex PCR Electrophoresis Fluorescent probes (FRET) Hybridization Microarrays Bead-based arrays Mass spectrometry Sequencing Spatial multiplexing Abbott PLEX-ID

25 Multiplex PCR Electrophoresis Fluorescent probes (FRET) Hybridization Microarrays Bead-based arrays Mass spectroscopy detection Sequencing Spatial multiplexing Life Technologies Ion Torrent Illumina MiSeq Roche GS Junior

26 Multiplex PCR Electrophoresis Fluorescent probes (FRET) Hybridization Microarrays Bead-based arrays Mass spectroscopy detection Sequencing Spatial multiplexing Idaho Technology FilmArray Life Technologies OpenArray

27 LAMP Loop mediated isothermal amplification Precipitation of pyrophosphates released during DNA amplification Visible turbidity Can modify to produce color or fluorescence Inexpensive No thermal cycler Visible to naked eye

28 Nucleic Acid Lateral Flow Simple, fast, cheap potential for developing world POC diagnostics May incorporate target amplification or signal amplification to increase sensitivity detection using enzymes, quantum dots, colloidal gold, etc. Future devices may incorporate integrated solid phase isothermal nucleic acid amplification

29 Which is the best one for my lab?

30

31 Technical Challenges of Multiplexing Nucleic acid extraction Loss of sensitivity Mispriming Cross-hybridization Competition

32 Expensive

33 ** Not for Diagnostic Testing **

34 Centralized Testing

35 Batch Testing

36 New Pathogens

37 Perpetuating the OldMicrobiology Education Budgets Laboratory infrastructure

38 Where are we headed?

39 The Future Integration of new point-of-care diagnostics onto wards Improved rapid serologic tests (antigen or antibody) Simple, rapid chip-based or lab-in-a-box molecular tests Real-time PCR, microarrays, single-molecular detection Target the most common pathogens Syndromic-based testing Expansion of clinical team Physicians, nurses, pharmacists plus laboratory technologists Operation and troubleshooting of advanced diagnostic BCCDC Public Health Microbiology & Reference Laboratory equipment

40 The Future Hospital laboratories Focus on rapid tests targeting common pathogens (multiplexing based on syndrome) More molecular tests, fewer culture-based tests Reference laboratories Traditional culture and phenotypic tests Maintenance of sequence databases to ensure molecular assays reflect the circulating strains of various organisms High volume tests Tests which do not require rapid turn-around times Advanced molecular tests Microarrays Metagenomics Genotyping and characterization of organisms BCCDC Public Health Microbiology & Reference Laboratory Whole genome sequencing

41 Point-of-Care Molecular Tests Integrated sample preparation Sealed lab-in-a-box systems Fast turn-around time High sensitivity Cepheid GeneXpert

42 Point-of-Care Molecular Tests Idaho Technologies FilmArray IQuum Liat Analyzer

43 Advancing the Molecular Revolution Revolution requires re-thinking of the entire system of delivering laboratory services Many new technologies emerging What is hype and what is the real thing? New technology inherently costs more How do we pay for it all? POC molecular tests are here How will we manage POC testing?

44 BCCDC Public Health Microbiology & Reference Laboratory Questions